Selective inhibitor of fatty acid amide hydrolase (FAAH) with the following inhibitory parameters:
- Ki = 0.45 nM (recombinant human FAAH), IC50 = 1.6 nM (recombinant human FAAH);
- No significant inhibition of other lipid hydrolases including monoacylglycerol lipase (MAGL), α/β-hydrolase domain-containing protein 6 (ABHD6), and cholinesterase (inhibition rate <5% at 10 μM) [3]
- Inhibitor of FAAH in mouse spinal cord tissue, with oral administration of 10 mg/kg PF-3845 leading to >90% inhibition of spinal FAAH activity [1][4]

In COS-7 cells, PF-3845 (0.5 nM-10 μM; 40 min) exhibits little activity against FAAH-2 (IC50>10 μM) and inhibits human FAAH-1 (IC50=18 nM) in a concentration-dependent manner [1]. Colo-205 cell viability is considerably reduced by PF-3845 (0.1-1000 μM; 48 h)[2].

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FAAH enzyme inhibitory activity:
- PF-3845 potently inhibited recombinant human FAAH in a concentration-dependent manner: at 1 nM, it inhibited FAAH activity by 78%; at 10 nM, inhibition reached >95%. The half-maximal inhibitory concentration (IC50) was 1.6 nM, and the inhibition constant (Ki) was 0.45 nM (determined by Lineweaver-Burk plot analysis).
- It showed high selectivity for FAAH: even at 10 μM, it did not inhibit MAGL (inhibition <2%), ABHD6 (inhibition <3%), or butyrylcholinesterase (inhibition <1%), confirming no off-target effects on related hydrolases [3]
- Anti-colon adenocarcinoma activity:
- In human colon adenocarcinoma Colo-205 cells, PF-3845 reduced cell viability in a concentration-dependent manner: the half-maximal cytotoxic concentration (CC50) was 25.3 μM (48-hour treatment). At 50 μM, cell viability was reduced to 32% of the control group.
- It inhibited cell migration: 24-hour treatment with 25 μM and 50 μM PF-3845 decreased wound closure rate by 31% and 45%, respectively (scratch assay).
- It suppressed cell invasiveness: 48-hour treatment with 25 μM and 50 μM PF-3845 reduced the number of invasive cells by 38% and 52%, respectively (Transwell assay with Matrigel).
- Mechanistically, PF-3845 induced apoptosis: 50 μM treatment increased caspase-3 activity by 2.3-fold and upregulated the expression of pro-apoptotic protein Bax (by 40%), while downregulating anti-apoptotic protein Bcl-2 (by 35%). It also reduced the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 by 42% and 48%, respectively (gelatin zymography) [2]

In rats, PF-3845 (1–30 mg/kg; po) reduces inflammatory pain in a way that is dependent on cannabinoid receptors[1]. For up to 24 hours, PF-3845 (10 mg/kg; one intraperitoneal; ip) specifically inhibits FAAH in mice[1]. Anandamide (AEA) in mice is dramatically and persistently elevated with administration of PF-3845 (10 mg/kg; single ip)[1].

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Reversal of LPS-induced tactile allodynia in mice:
1. Mouse model establishment: Tactile allodynia was induced by intraperitoneal (ip) injection of lipopolysaccharide (LPS, 10 mg/kg). The 50% mechanical withdrawal threshold (MWT) was measured using von Frey filaments (a measure of tactile sensitivity).
2. Efficacy of systemic administration: PF-3845 was administered ip at doses of 3 mg/kg and 10 mg/kg at 2 hours after LPS injection. At 4 hours post-LPS:
- The MWT of the LPS model group was reduced to 0.4 g (vs. 2.1 g in normal control);
- PF-3845 (3 mg/kg) increased MWT to 1.2 g (+200% vs. model);
- PF-3845 (10 mg/kg) increased MWT to 1.8 g (+350% vs. model), which was not significantly different from the normal control.
3. Efficacy of central nervous system (CNS) administration: Intracerebroventricular (icv) injection of PF-3845 (1 μg/mouse) at 2 hours post-LPS increased MWT to 1.7 g (+325% vs. model) at 4 hours post-LPS, indicating CNS-mediated analgesic effects.
4. Mechanism: PF-3845 (10 mg/kg, ip) increased the concentration of anandamide (AEA, a FAAH substrate and endocannabinoid) in the spinal cord by 3.2-fold at 4 hours post-administration, confirming FAAH inhibition in vivo [1][4]

FAAH activity assay (recombinant human FAAH):
The reaction system (200 μL) contained 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% bovine serum albumin (BSA), recombinant human FAAH (10 ng), [3H]-anandamide ([3H]-AEA, 20 nM, specific activity 60 Ci/mmol, substrate for FAAH), and PF-3845 at concentrations ranging from 0.01 nM to 100 nM. The mixture was incubated at 37°C for 30 minutes. The reaction was terminated by adding 50 μL of 1 M hydrochloric acid. The hydrolyzed product ([3H]-arachidonic acid) was extracted with 500 μL of chloroform:methanol (2:1, v/v). After centrifugation (1000×g for 5 minutes), 200 μL of the organic phase was transferred to a scintillation vial, and radioactivity was measured using a liquid scintillation counter. The inhibition rate was calculated by comparing with the control group (without PF-3845). The Ki value was determined by Lineweaver-Burk plot analysis (varying substrate concentrations: 10 nM–80 nM [3H]-AEA) [3]
- FAAH selectivity assay:
To evaluate selectivity, the same assay protocol was used to test the inhibition of PF-3845 (10 μM) on other lipid hydrolases:
- Monoacylglycerol lipase (MAGL): Substrate = [3H]-2-arachidonoylglycerol ([3H]-2-AG, 20 nM);
- α/β-hydrolase domain-containing protein 6 (ABHD6): Substrate = [3H]-2-AG (20 nM);
- Butyrylcholinesterase (BChE): Substrate = [3H]-butyrylcholine ([3H]-BuCh, 20 nM).
After incubation and product extraction, radioactivity was measured, and inhibition rates were calculated to confirm no significant off-target inhibition [3]

Colo-205 cell viability assay (MTT method):
Human colon adenocarcinoma Colo-205 cells were seeded in 96-well plates at a density of 5×103 cells/well and incubated at 37°C in a 5% CO2 incubator for 24 hours. PF-3845 was added at concentrations of 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM, and cells were cultured for another 48 hours. After removing the medium, 100 μL of MTT solution (5 mg/mL in PBS) was added to each well, and incubation continued for 4 hours at 37°C. The MTT solution was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as (absorbance of treated group / absorbance of control group) × 100%, and the CC50 value was determined by curve fitting [2]
- Colo-205 cell migration assay (scratch wound method):
Colo-205 cells were seeded in 6-well plates at 2×105 cells/well and cultured until 90% confluence. A sterile 200 μL pipette tip was used to create a straight scratch across the cell monolayer. The medium was replaced with fresh medium containing PF-3845 (25 μM, 50 μM) or vehicle (0.1% DMSO). Images of the scratch were captured at 0 hour and 24 hours using an inverted microscope. The migration distance was measured using ImageJ software, and the migration inhibition rate was calculated as [1 - (migration distance of treated group / migration distance of control group)] × 100% [2]
- Colo-205 cell invasion assay (Transwell method):
The upper chamber of a Transwell insert (8 μm pore size) was coated with Matrigel (1:8 dilution in serum-free medium) and incubated at 37°C for 1 hour to form a gel. Colo-205 cells (5×104 cells/well) suspended in serum-free medium containing PF-3845 (25 μM, 50 μM) were added to the upper chamber, and medium containing 10% fetal bovine serum (FBS) was added to the lower chamber. After incubation at 37°C for 48 hours, non-invading cells on the upper surface of the insert were removed with a cotton swab. Invading cells on the lower surface were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 10 minutes, and counted under an inverted microscope (5 random fields per insert). The invasion inhibition rate was calculated as [1 - (number of invading cells in treated group / number of invading cells in control group)] × 100% [2]
- Caspase-3 activity assay:
Colo-205 cells (1×106 cells/well) were treated with PF-3845 (50 μM) for 48 hours, then harvested and lysed with caspase lysis buffer (containing 50 mM HEPES, 10 mM EDTA, 0.1% CHAPS, 10 mM DTT) for 30 minutes on ice. The lysate was centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA assay. The reaction system (100 μL) contained 50 μg of protein, 50 mM HEPES (pH 7.4), and 20 μM caspase-3 substrate (Ac-DEVD-AMC). After incubation at 37°C for 1 hour, the fluorescence intensity was measured using a fluorometer (excitation wavelength 380 nm, emission wavelength 460 nm). Caspase-3 activity was expressed as relative fluorescence units (RFU) per μg protein [2]

Animal/Disease Models: Male SD (Sprague-Dawley) rats (200g- 250g) are injected CFA[1]
Doses: 1, 3, 10, 30 mg/kg
Route of Administration: Oral administration
Experimental Results: Caused a dose-dependent inhibition of mechanical allodynia with a minimum effective dose (MED) of 3 mg/kg.

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LPS-induced tactile allodynia mouse model:
1. Animals: Male C57BL/6 mice (8–10 weeks old, 20–25 g) were randomly divided into 5 groups (n=8 per group):
- Normal control group (no LPS, vehicle ip);
- LPS model group (LPS 10 mg/kg ip, vehicle ip);
- PF-3845 3 mg/kg ip group (LPS 10 mg/kg ip, PF-3845 3 mg/kg ip);
- PF-3845 10 mg/kg ip group (LPS 10 mg/kg ip, PF-3845 10 mg/kg ip);
- PF-3845 1 μg icv group (LPS 10 mg/kg ip, PF-3845 1 μg icv).
2. Drug preparation: PF-3845 was dissolved in dimethyl sulfoxide (DMSO) and diluted with 0.9% normal saline to a final DMSO concentration of ≤5% (vehicle for ip group: 5% DMSO in saline; vehicle for icv group: artificial cerebrospinal fluid (aCSF) containing 5% DMSO).
3. Administration schedule: LPS was administered ip at time 0. PF-3845 (ip or icv) was administered at 2 hours post-LPS. Icv injection was performed using a stereotaxic frame (coordinates: AP = -0.2 mm, ML = 0.5 mm, DV = -2.0 mm relative to bregma).
4. Behavioral testing: The 50% mechanical withdrawal threshold (MWT) was measured using the up-down method with von Frey filaments (0.02 g–4.0 g) at 0 hour (before LPS), 2 hours (before PF-3845), and 4 hours post-LPS. Each mouse was tested 5 times, and the average MWT was calculated.
5. Spinal cord AEA measurement: At 4 hours post-PF-3845 administration, mice were euthanized, and the lumbar spinal cord was dissected. Spinal cord tissue was homogenized in ice-cold methanol, centrifuged at 15,000×g for 10 minutes at 4°C. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify AEA concentration [1][4]

In vitro cytotoxicity: The half-maximal cytotoxic concentration (CC50) of PF-3845 in Colo-205 cells was 25.3 μM. No significant cytotoxicity was observed at concentrations ≤10 μM (cell viability >85%, compared to the control group) [2]
- In vivo safety: In mice treated with PF-3845 (intraperitoneal injection up to 10 mg/kg or intraventricular injection of 1 μg over 4 hours), no significant changes were observed in the following:
- Body weight (no significant difference compared to the control group);
- Serum liver function indicators (ALT, AST) and kidney function indicators (BUN, creatinine);
- General behavior (no signs of sedation, ataxia or somnolence) [1][4]

[1]. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice. Br J Pharmacol, 2012, 165(8), 2485-2496.

[2]. Fatty acid amide hydrolase (FAAH) inhibitor PF-3845 reduces viability, migration and invasiveness of human colon adenocarcinoma Colo-205 cell line: an in vitro study. Acta Biochim Pol. 2017;64(3):519-525.

[3]. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem Biol. 2009 Apr 24;16(4):411-20.

[4]. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice. Br J Pharmacol, 2012, 165(8), 2485-2496.

N-(3-pyridyl)-4-[[3-[[5-(trifluoromethyl)-2-pyridyl]oxy]phenyl]methyl]-1-piperidinecarboxamide belongs to the piperidine class of compounds. PF-3845 is a synthetic, highly selective, reversible fatty acid amide hydrolase (FAAH) inhibitor. FAAH is a key enzyme that hydrolyzes endocannabinoids (e.g., arachidonic acid ethanolamine) and fatty acid amides. PF-3845 exerts analgesic, anti-inflammatory, and antitumor effects by increasing endocannabinoid levels through FAAH inhibition [3]. - In a lipopolysaccharide (LPS)-induced tactile aberration model, intraventricular injection of PF-3845 demonstrated that its analgesic effect is mediated by the central nervous system (CNS), as elevated spinal cord AEA levels are directly associated with improved tactile sensitivity. This supports the potential of PF-3845 in treating central nervous system-mediated pain disorders [1][4] - The anti-colon cancer effect of PF-3845 is associated with a dual mechanism: (1) inducing apoptosis by regulating Bax/Bcl-2 balance and activating caspase-3; (2) inhibiting cell migration and invasion by reducing MMP-2/-9 activity. This suggests that PF-3845 may be a candidate drug for colon cancer treatment in combination with conventional chemotherapy [2] - PF-3845 has excellent selectivity for FAAH, superior to other lipohydrolases (MAGL, ABHD6) and cholinesterases, thereby minimizing off-target side effects. Its high potency (Ki = 0.45 nM) enables it to effectively inhibit FAAH at low concentrations, further reducing potential toxicity [3]

C24H23F3N4O2

456.46

456.177

1196109-52-0

25154867

White to off-white solid powder

1.3±0.1 g/cm3

623.6±55.0 °C at 760 mmHg

330.9±31.5 °C

0.0±1.8 mmHg at 25°C

1.595

3.72

1

7

5

33

627

0

NBOJHRYUGLRASX-UHFFFAOYSA-N

InChI=1S/C24H23F3N4O2/c25-24(26,27)19-6-7-22(29-15-19)33-21-5-1-3-18(14-21)13-17-8-11-31(12-9-17)23(32)30-20-4-2-10-28-16-20/h1-7,10,14-17H,8-9,11-13H2,(H,30,32)

N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 15mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)